Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*

Protocol summary

1. Prepare cells (0.5 - 1 × 10⁶ cells/mL)
2. Add 1 µL 500X Nitrixyte™ Orange into 0.5 mL cell suspension
3. Incubate cells with test compounds and Nitrixyte™ Orange at 37°C
4. Analyze with a flow cytometer

Important notes
Thaw all the components at room temperature before use.

1. NONOate Positive Control stock solution (50 mM):
Add 200 µL of ddH₂O into the vial of NONOate Positive Control (Component B) to make 50 mM stock solution.

1. NONOate Positive Control working solution
Dilute the NONOate Positive Control stock solution to a 1 - 2 mM working solution with Assay Buffer (Component C).

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Add 1 µL of 500X Nitrixyte™ Orange (Component A) into 0.5 mL cell suspension. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with Nitrixyte™ Orange.
   
   
2. Incubate cells with test compounds and Nitrixyte™ Orange at 37°C for a desired period of time to generate endogenous or exogenous NO. Note: The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment. Note: We have used Raw 264.7 cells incubated with 1X Nitrixyte™ Orange, 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) in cell culture medium at 37°C for 16 hours.
   
   
3. Spin down cells that have pre-incubated with Nitrixyte™ Orange for 30 minutes. Resuspend cells with 1 mM DEA NONOate positive control working solution, and incubate at 37°C for another 30 minutes. See Figure 1 for details.
   
   
4. Monitor the fluorescence intensity at the FL2 channel (Ex/Em = 488/590 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.